The long-term objective of the investigator's laboratory is to understand the role of epithelial sodium (Na +) channels (ENaC) in regulating Na + homeostasis and blood pressure in normal and disease states. The overall design of this project is to test the hypothesis that ENaC function is regulated by Nedd4, an ubiquitin protein ligase, and more specifically we hypothesize that ablation of the interaction between ENaC and Nedd4 in the renal cortical collecting duct (CCD) will lead to inappropriate Na + absorption, disrupted Na+/K + homeostasis, and high blood pressure. These studies are designed to elucidate the mechanism(s) of ENaC interactions with regulatory proteins through tissue-specific inactivation of gene function. To accomplish this goal we will pursue the following specific aims. Aim 1. To introduce loxP sites into the mouse Nedd4-2 gene, one of the two members in the Nedd4 family and the one shown to be interacting with ENaC. Using gene targeting via homologous recombination in mouse ES cells, we will introduce loxP sites that will flank a region of the mouse Nedd4-2 gene that is essential for its function. Aim 2. To generate mice expressing a tamoxifen-inducible Cre recombinase selectively in renal CCD. We will utilize the endogenous Aqp2 promoter to direct cell-specific expression of the Cre recombinase. This will also be accomplished in ES cells to insert the Cre transgene into the mouse Aqp2 locus. Aim 3. To generate mice with CCD-specific knockout of Nedd4-2 gene. Crossing the mouse lines generated in Aims 1 and 2, we will be able to generate mice homozygous for the floxed Nedd4-2 allele and heterozygous for the Cre transgene at the Aqp2 locus or mice with one floxed and one null allele and heterozygous for the Cre transgene at the Aqp2 locus. Deletion of the Nedd4-2 gene in renal CCD can be accomplished with the introduction of tamoxifen in a temporally regulated fashion. Aim 4. Detailed analyses, from molecular biology, cellular biology, electrophysiology, to whole animal physiology, will be carried out with these mice to test our hypothesis, and to assess the physiological consequences of the deletion with regard to Na + homeostasis and blood pressure regulation. In addition, upon completion of this study we will provide two important lines of mice for the research community: a line of CCD-specific, inducible Cre recombinase mice for the analysis of other genes expressed in CCD, and a line of mice carrying a floxed Nedd4-2 allele for studies of Nedd4 family regulation of protein degradation in other organs or tissues. It is anticipated that these studies will provide new insights into the interaction of ENaC with regulatory proteins, Nedd4-2 in particular, and on the role of ENaC regulation of Na + absorption by the CCD. Furthermore, it is anticipated that these studies will provide new information on other ways that Na + transport can be regulated, and these might be relevant to disorders of Na + balance that cause hypertension, such as Liddle's syndrome.